Despite research efforts over the past two decades, glioblastoma (GBM) is still a devastating, deadly cancer with a median overall survival of less than 15 months. Oncolytic virotherapy is a promising approach for GBM treatment because it not only involves direct lysis of tumor cells and spares normal cells but also boosts immune responses to tumor cells. However, early clearance of oncolytic viruses by innate immune responses, poor virus propagation in tumor cells, and insufficient viral entry and/or spread present barriers that limit the efficacy of oncolytic virotherapy. In this application, we generated oncolytic herpes simplex virus 1 (oHSV) carrying a ?don't eat me signal?, i.e., being engineered to express a ligand, E-cadherin, of the inhibitory receptor of natural killer (NK) cells, KLRG1. This next-generation oHSV carrying E-cadherin (named E-oHSV) reduces the deleterious NK-mediated clearance of oHSV. Importantly, unlike the drugs that we and others previously used to suppress oncolytic virus clearance by innate immune responses, the novel E-oHSV that we generated should not have an immunosuppressive effect systemically or even in the tumor (GBM) microenvironment. That is, E-oHSV only reduces activation of NK cells that bind to or attack E-oHSV-infected cells in the tumor microenvironment, while the remaining NK cells will maintain their potency against GBM cells. E-oHSV also showed enhanced viral production likely by increasing cell-to-cell fusion between virus- infected and uninfected cells. GBM cells do not have endogenous E-cadherin expression; however, interestingly, we found that E-cadherin is not only highly expressed on the surface of E-oHSV-infected cells but also loaded on free viral particles, the latter of which facilitates viral entry and/or spread in solid tumors. These improvements on oncolytic viruses led to significantly enhanced survival of mice bearing patient-derived GBM cells and also prolonged animal survival in an orthotopic, immunocompetent GBM model. In this application, we propose to mechanistically characterize this E-oHSV and to strengthen our hypothesis that a next- generation oHSV expressing E-cadherin, E-oHSV, plays multiple roles to overcome the barriers of oncolytic virotherapy and thus has a superior efficacy for GBM treatment. Three aims have been proposed: Aim 1 is to explore the mechanisms by which innate immune responses to E-oHSV are inhibited. Aim 2 is to explore the mechanisms by which E-oHSV enhances cell-to-cell infection, viral binding, and thus viral spread and distinguish them from the NK inhibition mechanism. Aim 3 is to investigate in vivo mechanisms, efficacy, and potential toxicity of E-oHSV for GBM treatment. Collectively, our study will not only address fundamental questions on NK cell and OV biology, but will also develop novel therapeutics for GBM treatment. It may also provide a platform to enhance efficacy of non-HSV oncolytic viruses.